METAL OXIDES AND/OR HYDRATES THEREOF FOR STABILIZING AN AQUEOUS PREPARATION AGAINST MICROBIAL GROWTH

Abstract

A process is described for stabilizing an aqueous preparation against microbial growth. Also described, is the aqueous preparation stabilized against microbial growth as well as the use of a source of at least one metal oxide and/or its hydrated form for reducing the amount of an antimicrobial agent against at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold in an aqueous preparation. The use of a composition including at least one antimicrobial agent and a source of at least one metal oxide and/or its hydrated form as an antimicrobial composition in an aqueous preparation, is also described. In addition, the use of magnesium oxide, magnesium hydroxide, calcium oxide, calcium hydroxide, half burnt dolomite, burnt dolomite, beryllium oxide, beryllium hydroxide, strontium oxide, strontium hydroxide, barium oxide, barium hydroxide, and mixtures thereof for increasing the pH of an aqueous preparation to a pH of above 8 and the use of an aqueous preparation stabilized against microbial growth in paper, plastics, polymer compositions, paint, coatings, concrete and/or agriculture applications are described.

Claims

1. A process for stabilizing an aqueous preparation against microbial growth, the process comprising the steps of: a) providing an aqueous preparation, optionally comprising at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold; b) providing at least one antimicrobial agent; c) providing a source of at least one metal oxide and/or its hydrated form; d) contacting the aqueous preparation of step a) with the at least one antimicrobial agent of step b); and e) contacting the aqueous preparation of step a) before and/or during and/or after step d) with the source of at least one metal oxide and/or its hydrated form such that the total amount of the at least one metal oxide and/or its hydrated form in the aqueous preparation is at least 100 ppm, calculated relative to the weight of water in the preparation.

2. The process according to claim 1, wherein the aqueous preparation of step a) comprises (i) at least one inorganic particulate material, and/or (ii) at least one organic material.

3. The process according to claim 1, wherein the aqueous preparation of (i) step a) and/or of step d) has a pH value of from 2 to 12, and/or (ii) step e) has a pH value of above 8, and/or (iii) step a) and/or of step d) and/or of step e) has a solids content of up to 85.0 wt.-% based on the total weight of the aqueous preparation.

4. The process according to claim 1, wherein the at least one strain of bacteria is selected from the group consisting of gram-negative bacteria, gram-positive bacteria and mixtures thereof.

5. The process according to claim 1, wherein (i) the at least one strain of bacteria is selected from the group consisting of Methylobacterium sp., Salmonella sp., Escherichia sp., Shigella sp., Enterobacter sp., Pseudomonas sp., Burkholderia sp., Bdellovibrio sp., Agrobacterium sp., Alcaligenes sp., Flavobacterium sp., Ochrobactrum sp., Kocuria sp., Rhizobium sp., Sphingobacterium sp., Aeromonas sp., Chromobacterium sp., Vibrio sp., Hyphomicrobium sp., Leptothrix sp., Micrococcus sp., Staphylococcus sp., Agromyces sp., Acidovorax sp., Commomonas sp., Brevundimonas sp., Spingobium sp., Thauera sp., Caldimonas sp., Hdrogenophaga sp., Teipidomonas sp., and mixtures thereof, and mixtures thereof, and/or (ii) the at least one strain of yeast is selected from the group comprising Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Microbotryomycetes, Candida sp., Yarrowia sp., Cryptococcus sp., Zygosaccharomyces sp., Rhodotorula sp., Saccharomyces sp., Pichia sp. and mixtures thereof, and/or (iv) the at least one strain of mold is selected from the group consisting of Acremonium sp., Alternaria sp., Aspergillus sp., Cladosporium sp., Fusarium sp., Mucor sp., Penicillium sp., Rhizopus sp., Stachybotrys sp., Trichoderma sp., Dematiaceae sp., Phoma sp., Eurotium sp., Scopulariopsis sp., Aureobasidium sp., Monilia sp., Botrytis sp., Stemphylium sp., Chaetomium sp., Mycelia sp., Neurospora sp., Ulocladium sp., Paecilomyces sp., Wallemia sp., Curvularia sp., and mixtures thereof.

6. The process according to claim 1, wherein the at least one antimicrobial agent of step b) is effective against the at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold when present in the aqueous preparation or the at least one antimicrobial agent of step b) is effective against the at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold when present in the aqueous preparation in the presence of the source of at least one metal oxide and/or its hydrated form.

7. The process according to claim 1, wherein the at least one antimicrobial agent of step b) is selected from the group consisting of phenols, halogenated phenols, halogen-containing compounds, halogen-releasing compounds, isothiazolinones, aldehyde-containing compounds, aldehyde-releasing compounds, guanidines, sulphones, thiocyanates, pyrithiones, antibiotics, quaternary ammonium salts, peroxides, perchlorates, amides, amines, heavy metals, biocidal enzymes, biocidal polypeptides, azoles, carbamates, glyphosates, sulphonamides and mixtures thereof.

8. The process according to claim 1, wherein the source of at least one metal oxide and/or its hydrated form comprises an oxide and/or a hydrated form of an element of group 2 of the periodic table.

9. The process according to claim 1, wherein the source of at least one metal oxide and/or its hydrated form is selected from the group consisting of magnesium oxide, magnesium hydroxide, calcium oxide, calcium hydroxide, half burnt dolomite, burnt dolomite, beryllium oxide, beryllium hydroxide, strontium oxide, strontium hydroxide, barium oxide, barium hydroxide and mixtures thereof.

10. The process according to claim 1, wherein step e) is carried out by adding the source of at least one metal oxide and/or its hydrated form to the aqueous preparation in an amount such that the total amount of the at least one metal oxide and/or its hydrated form in the aqueous preparation is from 100 ppm to 5,000 ppm, calculated relative to the weight of water in the preparation.

11. The process according to claim 1, wherein step e) is carried out before step d).

12. The process according to claim 1, wherein step d) and/or step e) is/are repeated one or more times.

13. The process according to claim 1, wherein step d) is carried out by adding the at least one antimicrobial agent to the aqueous preparation (i) in an amount being at least 10% below the minimum inhibitory concentration (MIC) of the at least one antimicrobial agent determined in the absence of the metal oxide and/or its hydrated form for the at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold, and/or (ii) in an amount such that the total amount of the at least one antimicrobial agent in the aqueous preparation is from 0.5 ppm to 6,000 ppm, calculated relative to the weight of water in the aqueous preparation.

14. An aqueous preparation stabilized against microbial growth obtained by the process according to claim 1.

15. An aqueous preparation stabilized against microbial growth, the preparation comprising a) at least one antimicrobial agent in an amount such that the total amount of the at least one antimicrobial agent in the aqueous preparation is from 0.5 ppm to 6,000 ppm, calculated relative to the weight of water in the aqueous preparation, and b) a source of at least one metal oxide and/or its hydrated form such that the total amount of the at least one metal oxide and/or its hydrated form in the aqueous preparation is at least 100 ppm, calculated relative to the weight of water in the aqueous preparation.

16. A method of reducing an amount of an antimicrobial agent against at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold, the method comprising providing a source of at least one metal oxide and/or its hydrated form in the aqueous preparation of claim 1, to reduce the amount of the antimicrobial agent against the at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold, wherein the amount of the antimicrobial agent in the aqueous preparation is at least 10% below the minimum inhibitory concentration (MIC) of the at least one antimicrobial agent, the MIC being determined in absence of the metal oxide and/or its hydrated form for the at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold.

17. A stabilized aqueous preparation, the preparation comprising an effective amount of an antimicrobial composition comprising a) at least one antimicrobial agent in an amount such that the total amount of the at least one antimicrobial agent in the aqueous preparation is from 0.5 ppm to 6,000 ppm, calculated relative to the weight of water in the aqueous preparation, and b) a source of at least one metal oxide and/or its hydrated form such that the total amount of the at least one metal oxide and/or its hydrated form in the aqueous preparation is at least 100 ppm, calculated relative to the weight of water in the aqueous preparation, wherein the preparation optionally comprises at least one strain of bacteria and/or at least one strain of yeast and/or at least one strain of mold.

18. A method of increasing pH of an aqueous preparation, the method of comprising adding an effective amount of magnesium oxide, magnesium hydroxide, calcium oxide, calcium hydroxide, half burnt dolomite, burnt dolomite, beryllium oxide, beryllium hydroxide, strontium oxide, strontium hydroxide, barium oxide, barium hydroxide and mixtures thereof to increase the pH of the aqueous preparation, which is a preparation as defined in claim 1, to a pH of above 8.

19. A method of stabilizing microbial growth in paper, plastics, polymer compositions, paint, coatings, concrete and/or agriculture applications, the method comprising adding an effective amount of the aqueous preparation of claim 14 to the paper, plastics, polymer compositions, paint, coatings, concrete and/or agriculture applications.

20. The process according to claim 2, wherein the at least one inorganic particulate material is selected from the group consisting of natural ground calcium carbonate, natural and/or synthetic precipitated calcium carbonate, surface-modified calcium carbonate, dolomite, kaolin, talcum, aluminium hydroxide, aluminium silicate, titanium dioxide, barium sulphate, hydroxyapatite and mixtures.

21. The process according to claim 2, wherein the at least one inorganic particulate material is natural ground calcium carbonate and/or synthetic precipitated calcium carbonate.

22. The process according to claim 2, wherein the at least one organic material is a carbohydrate.

23. The process according to claim 22, wherein the carbohydrate is selected from the group consisting of starch, sugar, cellulose, cellulose based pulp, glycerol, hydrocarbons and mixtures thereof.

24. The process according to claim 3, wherein the pH value in step a) and/or step d) is from 6 to 11.5.

25. The process according to claim 3, wherein the pH value in step a) and/or step d) is from 7 to 10.5.

26. The process according to claim 3, wherein the pH value in step e) is above 9.

27. The process according to claim 3, wherein the solids content of step a) and/or step e) is from 10.0 wt.-% to 82.0 wt.-%.

28. The process according to claim 3, wherein the solids content of step a) and/or step e) is from 20.0 wt.-% to 80.0 wt.-%.

29. The process according to claim 5, wherein for the at least one strain of bacteria, one or more of the following apply: the Escherichia sp. is Escherichia coli; the Pseudomonas sp. is Pseudomonas mendocina, Pseudomonas stutzeri, Pseudomonas aeruginosa, and/or Pseudomonas putida; the Burkholderia sp. is Burkholderia cepacia; the Alcaligenes sp. is Alcaligenes faecalis; the Ochrobactrum sp. is Ochrobactrum tritici; the Kocuria sp. is Kocuria rhizophila; the Rhizobium sp. is Rhizobium radiobacter; the Staphylococcus sp. is Staphylococcus aureus; the Commomonas sp. is Commmonas aquatic; the Spingobium sp. is Spingobium yanoikuyae; and the Thauera sp. is Thauera mechernichensis.

30. The process according to claim 5, wherein for at least one strain of yeast, one or more of the following apply: the Candida sp. is Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida guilliermondii, Candida viswanathii, Candida lusitaniae or a mixture thereof; the Yarrowia sp. is Yarrowia lipolytica; the Cryptococcus sp. is Cryptococcus gattii or Cryptococcus neofarmans; the Rhodotorula sp. is Rhodotorula mucilaginosa; the Saccharomyces sp. is Saccharomyces cerevisiae; and the Pichia sp. is Pichia membranifaciens.

31. The process according to claim 7, wherein when the at least one antimicrobial agent is an antibiotic, the antibiotic is a -lactam antibiotic.

32. The process according to claim 9, wherein the source of at least one metal oxide and/or its hydrated form is magnesium oxide and/or magnesium hydroxide.

33. The process according to claim 13, wherein the at least one antimicrobial agent is added to the aqueous preparation in an amount being at least 25%.

34. The process according to claim 13, wherein the at least one antimicrobial agent is added to the aqueous preparation in an amount being at least 50%.

35. The process according to claim 13, wherein the at least one antimicrobial agent is added to the aqueous preparation in an amount being at least 75%.

36. The method according to claim 16, wherein the source of at least one metal oxide and/or its hydrated form is selected from the group consisting of magnesium oxide, magnesium hydroxide, calcium oxide, calcium hydroxide, half burnt dolomite, burnt dolomite, beryllium oxide, beryllium hydroxide, strontium oxide, strontium hydroxide, barium oxide, barium hydroxide, and mixtures thereof.

37. The method according to claim 16, wherein the source of at least one metal oxide and/or its hydrated form is magnesium and/or magnesium hydroxide.

38. The method according to claim 16, wherein the amount of the antimicrobial agent is at least 25% below the MIC.

39. The method according to claim 16, wherein the amount of the antimicrobial agent is at least 50% below the MIC.

40. The method according to claim 16, wherein the amount of the antimicrobial agent is at least 75% below the MIC.

41. The method according to claim 18, wherein the pH is increased by adding magnesium oxide and/or magnesium hydroxide.

42. The method according to claim 18, wherein the pH is above 9.

43. The method according to claim 42, wherein the pH is above 9.2.

44. The method according to claim 42, wherein the pH is above 9.6.

45. The method according to claim 42, wherein the pH is above 9.8.

46. The method according to claim 42, wherein the pH is above 10.

47. The method according to claim 42, wherein the pH is from 10 to 10.5.

Description

EXAMPLES

Measurement Methods

[0204] The following measurement methods are used to evaluate the parameters given in the description, examples and claims.

BET Specific Surface Area of a Material

[0205] The BET specific surface area was measured via the BET process according to ISO 9277 using nitrogen.

Particle Size Distribution (Mass % Particles with a Diameter <X) and Weight Median Diameter (d.sub.50) of a Particulate Material

[0206] Weight median grain diameter and grain diameter mass distribution of a particulate material were determined via the sedimentation process, i.e. an analysis of sedimentation behaviour in a gravitational field. The measurement was made with a Sedigraph 5100 of Micromeritics Instrument Corporation.

[0207] The method and the instrument are known to the skilled person and are commonly used to determine grain size of fillers and pigments. The measurement is carried out in an aqueous solution of 0.1 wt.-% Na.sub.4P.sub.2O.sub.7. The samples are dispersed using a high speed stirrer and supersonics.

pH Measurement

[0208] The pH of the water samples is measured by using a standard pH-meter at approximately 25 C.

Brookfield-Viscosity

[0209] All Brookfield-viscosities are measured with a Brookfield DV-II Viscometer equipped with a LV-3 spindle at a speed of 100 rpm and room temperature (203 C.).

Amount of Additives

[0210] Unless otherwise stated, all amounts quoted in ppm represent mg values per kilogram of water in the aqueous preparation. Concentrations are further quoted in mmol/kg (millimol per kilogram) or mol/l (mol per litre) according to the International System of Units in the water of the aqueous preparation.

Bacterial Count

[0211] All quoted bacterial counts in the Tables here below are either cfu/ml (colony forming units per millilitre) or cfu/plate (colony forming units per plate) wherein cfu/ml were determined after 2-5 days following plate-out and in accordance with counting method described in Bestimmung von aeroben mesophilen Keimen, Schweizerisches Lebensmittelbuch, chapter 56, section 7.01, edition of 1985, revised version of 1988. Unless otherwise stated, per tryptic soy agar plate (TSA, prepared using BD 236950) 0.1 ml of a 1:10 dilution in phosphate buffered saline (PBS; pH=7.4, 137 mmol/l NaCl, 2.7 mmol/l KCl, 10 mmol/l Na.sub.2HPO.sub.4, 1.8 mmol/l KH.sub.2PO.sub.4) was plated. Counts from 10 000 cfu/ml to 99 999 cfu/ml are reported as >10 000 cfu/ml. Counts of 100 000 cfu/ml and above are reported as >100 000 cfu/ml. The counting method for cfu/plate was as follows. The aqueous preparations were stirred well with a cotton swap (e.g. Applimed SA, No. 1102245); the excess aqueous preparation was removed by dipping it gently to the side of the aqueous preparation container, leaving approximately 200 mg of aqueous preparation on the swap. Then three even streaks were made on a tryptic soy agar plate (TSA, prepared using BD 236950) from right to left and three more from top to bottom. TSA plates were then incubated for 48 h at 30 C. Colony forming units (cfu) were then counted and reported as cfu/plate. Counts from 100 to 999 cfu per plate are reported as >100 cfu/plate. Counts of 1 000 cfu and above per plate are reported as >1 000 cfu/plate.

Solids Content

[0212] The solids content is measured using a Moisture Analyzer of Mettler-Toledo MJ33. The method and the instrument are known to the skilled person.

Preparation of Bacteria

[0213] Fresh bacteria cultures of the bacteria were prepared by dilution streaking onto a tryptic soy agar plate (TSA, Becton Dickinson no. 236950) and incubation for 16 to 20 h at 35 C. The bacteria were either environmental clonal isolates from calcium carbonate slurries, identified by 16S rRNA sequencing as Comomonas aquatic, Pseudomonas stutzeri, Thauera mechernichensis, Brevundimonas, Rhizobium radiobacter, Spingobium yanoikuyae, Caldimonas sp., Hydrogenophaga sp., and/or the commercially available bacteria Escherichia coli DSM 1576 and Staphylococcus aureus strains DSM 346.

[0214] For the preparation of an overnight culture of bacterium species, fresh bacteria were used to inoculate separately 3-5 ml TSB (Fluka 22092). The inoculated TSB was incubated for 16 to 20 h at 30 C. with agitation at 150 rotations per minutes (rpm) For the preparation of a fresh adapted culture of a single bacterium species, 50 g calcium carbonate slurry were inoculated with 0.2 ml overnight culture and incubated for 72 h at 30 C.

[0215] For the preparation of an adapted mixture culture consisting of multiple species, fresh adapted cultures of a single bacterium species were combined at equal ratios.

Minimum Inhibitory Concentration (MIC)

[0216] For determining the MIC, the tested microorganism, i.e. the strain of bacteria and/or strain of yeast and/or strain of mould, were freshly grown until the end of the logarithmic growth phase according to the requirements of the individual species to a density of approximately 10.sup.7-10.sup.9 cells/ml.

[0217] For example, fresh bacteria cultures of the bacteria E. coli, e.g. E. coli DSM 1576, and S. aureus, e.g. S. aureus strains DSMZ 346, were prepared by inoculation of 3 ml liquid growth media (tryptic soy broth, e.g. Fluka Cat. No. 22092) from a stock culture and incubation for 16 to 20 h at 30 C. with agitation at 150 rotations per minutes (rpm) leading to a cell density of approximately 210.sup.8 cells/ml.

[0218] Metal oxide and/or its hydrated form were added to the aqueous preparation (e.g. CaCO.sub.3 slurry) as indicated.

[0219] The antimicrobial agent to be tested was added to the aqueous preparation, e.g. CaCO.sub.3 slurry, with or without metal oxide in increasing concentrations starting from 0 ppm (=without antimicrobial agent). The concentrations ranged from concentrations higher than recommended by the supplier, to very low amounts (as low as 1 ppm or less). Of each antimicrobial agent concentration a 3 ml sample of aqueous preparation was combined with 20 l fresh bacterial culture.

[0220] If a curative MIC was tested, the bacteria were added to the aqueous preparation before the antimicrobial agent and the metal oxide and/or its hydrated form was added, using 20 l of a fresh bacterial culture per 3 ml of aqueous preparation or 0.1 ml of a fresh adapted culture from a slurry per 3 ml of aqueous preparation.

[0221] All samples were incubated at 30 C. for 24 h. After incubation colony forming units (cfu) per plate (cfu/plate) were determined as described above under bacterial count.

[0222] The MIC for bacteria is defined as the lowest concentration of antimicrobial agent in the presence or absence of metal oxide and/or its hydrated form amongst all samples tested, where the bacterial concentration dropped below 100 cfu/plate. The MIC for yeast and mould is defined as the lowest concentration of antimicrobial agent in the presence or absence of metal oxide and/or its hydrated form amongst all samples tested, where the microbial concentration dropped below 20 cfu/plate. The test was valid only, if the sample without antimicrobial agent showed more than 100 cfu/plate for bacteria and more than 20 cfu/plate for yeast and mould. If none of the samples containing the respective antimicrobial agent dropped below 100 cfu/plate for bacteria and below 20 cfu/plate for yeast and mould the MIC was reported as >highest antimicrobial agent concentration tested (e.g. >1 000 ppm).

Example 1: Characterization of Inorganic Particulate Materials

[0223] The composition of different inorganic particulate materials was determined by X-ray diffraction (XRD) analysis known to the person skilled. Briefly, samples were analysed with a Bruker D8 Advance powder diffractometer obeying Bragg's law. This diffractometer consists of a 2.2 kW X-ray tube, a sample holder, a - goniometer, and a VNTEC-1 detector. Nickel-filtered Cu K radiation was employed in all experiments. The profiles were chart recorded automatically using a scan speed of 0.7 per minute in 2 . The resulting powder diffraction pattern was used to classify the material contents using appropriate software and databases. In this study the DIFFRACsuite software packages EVA and SEARCH, based on reference patterns of the ICDD PDF 2 database were used. Quantitative calculation was carried out with EVA software calculating the quantitative amount relative to the peak to background area.

[0224] The burnt dolomite consisted of 1.9 wt.-% calcite (crystalline form of calcium carbonate, CaCO.sub.3), 1.0 wt.-% dolomite (crystalline CaMg(CO.sub.3).sub.2), 55.2 wt.-% lime (calcium oxide, CaO), 38.7 wt.-% periclase (magnesium oxide, MgO), 0.3 wt.-% quarz (SiO.sub.2), 2.6 wt.-% anhydrite/bassanite (CaSO.sub.4, /CaSO.sub.4 0.5H.sub.2O) and 0.2 wt.-% maghemite (Fe.sub.2O.sub.3). All wt.-% are based on the total dry weight of the burnt dolomite.

[0225] The half burnt dolomite consisted of 70.7 wt.-% calcite (crystalline form of calcium carbonate, CaCO.sub.3), 0.5 wt.-% dolomite (crystalline CaMg(CO.sub.3).sub.2) and 28.8 wt.-% periclase (magnesium oxide, MgO). All wt.-% are based on the total dry weight of the half burnt dolomite.

[0226] Pure MgO (CAS No 1309-48-4) had a purity of 99.7 wt.-% and a BET specific surface area of 186 m.sup.2/g and a median particle size of d.sub.50 2.44 m.

Example 2: Preparation of Calcium Carbonate Slurries

[0227] Unless otherwise stated, the calcium carbonate slurries used had solid contents in the range from 75 to 78 wt.-%, based on the total weight of the slurries. The solids of the slurries consisted of 86.5 wt.-% calcium carbonate and 13.5 wt.-% dolomite (crystalline CaMg(CO.sub.3).sub.2, corresponding to 6.17 wt.-% MgCO.sub.3), based on the total weight of the solids, as determined by XRD analysis.

[0228] These aqueous slurries of calcium carbonate (Austrian marble; d.sub.50=10 m; 21%<2 m) can be prepared as follows. The slurry was wet ground at 95 C. using 0.6 wt.-% in respect to dry solids material of a sodium/calcium neutralised polyacrylate grinding agent (Mw 6 000) in a 200 l vertical ball mill to a final particle size distribution of d.sub.50=0.7 m; 90 wt.-%<2 m. For some slurries, additives, such as MgO or half burnt dolomite, were added prior grinded at 1 000 ppm based on dry weight and grinded together with the carbonates.

Example 3: Three-Challenge Preservation Test

[0229] The indicated amounts of ingredients were introduced into 50 g samples of calcium carbonate slurry (76.4 wt.-% solids content). As antimicrobial agent methylisothiazolinone (MIT, CAS No. 2682-20-4) was dosed as 20 wt.-% solution. Pure MgO was dosed from a suspension containing water and MgO with a purity of 99.7 wt.-% MgO as described in Example 1, based on the total mineral content at 4 mol MgO/l suspension (corresponds to 161.23 g MgO/ or 161.72 g minerals/l). Burnt dolomite with an MgO content of 38.7 wt.-% was dosed from a suspension at 2 mol MgO/l suspension (corresponds to 80.6 g MgO/l or 208.3 g burnt dolomite). The suspensions were mixed well immediately prior dosing to ensure an even distribution of the solids. Suspensions of MgO and burnt dolomite were added. In parallel, control samples of calcium carbonate slurry were prepared in absence of any additives. All samples were incubated to reach an equilibrium for 3 days at 30 C.

[0230] The samples were then inoculated three times with 1 mL of adapted mixture culture consisting of Comomonas aquatic, Pseudomonas stutzeri, Thauera mechernichensis, Brevundimonas intermedia, Rhizobium radiobacter, Spingobium yanoikuyae, Caldimonas sp., and Hydrogenophaga sp. After each inoculation, the samples were incubated at 30 C. for 24 hours. Thereafter, a 1:10 dilution of an aliquot was prepared in phosphate buffered saline (PBS; pH=7.4, 137 mmol/l NaCl, 2.7 mmol/1 KCl, 10 mmol/l Na.sub.2HPO.sub.4, 1.8 mmol/l KH.sub.2PO.sub.4) and was plated on TSA plates. These plates were incubated at 30 C. and analysed after 5 days. The first inoculation was performed 3 days, the second inoculation 5 days and the third inoculation 10 days after addition of ingredients.

[0231] The results of the inoculations are outlined in Table 1 below.

TABLE-US-00001 TABLE 1 Three-challenge Preservation test with a complex bacterial mixture as inoculum in slurry with different amounts of MgO, CaO and/or MIT as antimicrobial agent. Additives in ppm per aqueous content Counts after Inoculation Burnt [cfu/ml] MIT MgO.sup.1 CaO.sup.2 Dolomite.sup.3 First Second Third pH 1 1 050 1 389 1 981 3 588 <100 <100 <100 10.2 2 750 1 389 1 981 3 588 <100 <100 <100 10.3 3 450 1 389 1 981 3 588 <100 <100 <100 10.4 4 150 1 389 1 981 3 588 <100 <100 <100 10.5 5 0 1 389 1 981 3 588 >100 000 >100 000 >100 000 10.3 6 1 050 3 000 4 279 7 752 <100 <100 <100 10.6 7 750 3 000 4 279 7 752 <100 <100 <100 10.6 8 450 3 000 4 279 7 752 <100 <100 <100 10.6 9 150 3 000 4 279 7 752 <100 <100 <100 10.8 10 0 3 000 4 279 7 752 >10 000 >100 000 >100 000 10.7 11 1 050 3 000 0 0 <100 <100 <100 9.9 12 750 3 000 0 0 <100 <100 <100 9.9 13 450 3 000 0 0 <100 <100 <100 10.1 14 150 3 000 0 0 <100 200 <100 10.0 15 0 3 000 0 0 >100 000 >100 000 >100 000 10.0 16 1 050 0 0 0 4 900 2 000 <100 8.5 17 750 0 0 0 >10 000 4 600 100 8.4 18 450 0 0 0 >10 000 >10 000 1 100 8.5 19 150 0 0 0 >100 000 >100 000 >10 000 8.7 20 0 0 0 0 >100 000 >100 000 >100 000 8.1 .sup.1Amount of MgO dosed via the burnt dolomite or the pure MgO. .sup.2Amount of CaO dosed via the burnt dolomite. .sup.3Amount of burnt dolomite added to reach the indicated MgO and CaO concentration.

Example 4: Three-Challenge Preservation Test

[0232] The indicated amounts of ingredients were introduced into 50 g samples of calcium carbonate slurry (77.1 wt.-% solids content). As antimicrobial agent methylisothiazolinone (MIT, CAS No. 2682-20-4) was dosed as 20% (w/w) solution. Pure MgO was dosed from a suspension containing water and MgO with a purity of 99.7 wt.-% MgO based on the total mineral content at 4 mol MgO/l suspension (corresponds to 161.24 g MgO/ or 161.73 g minerals/l). In parallel, control samples of calcium carbonate slurry were prepared in absence of any additives. All samples were incubated to reach an equilibrium for 3 days at 30 C.

[0233] All samples were then inoculated three times with 1 mL of adapted mixture culture consisting of Comomonas aquatic, Pseudomonas stutzeri, Thauera mechernichensis, Brevundimonas intermedia, Rhizobium radiobacter, Spingobium yanoikuyae, Caldimonas sp., and Hydrogenophaga sp. After each inoculation, the samples were incubated at 30 C. for 24 hours. Thereafter, a 1:10 dilution of an aliquot was prepared in phosphate buffered saline (PBS) and was plated on TSA plates. These plates were incubated at 30 C. and analysed after 5 days. The first inoculation was performed 3 days, the second inoculation 5 days and the third inoculation 10 days after addition of ingredients.

[0234] The results of the inoculations are outlined in Table 2 below.

TABLE-US-00002 TABLE 2 Three-challenge Preservation test with a complex bacterial mixture as inoculum in slurry with different amounts of MgO and/or MIT as antimicrobial agent. Additives in ppm per aqueous Counts after Inoculation content [cfu/ml] MIT MgO.sup.1 First Second Third pH 1 750 3 000 <100 100 <100 10.06 2 375 3 000 200 <100 400 10.11 3 187.5 3 000 <100 <100 <100 10.09 4 0 3 000 >100 000 >100 000 >100 000 10.05 5 750 0 >10 000 5 400 5 300 8.58 6 375 0 >100 000 >10 000 >10 000 8.6 7 187.5 0 >100 000 >10 000 >10 000 8.69 8 0 0 >100 000 >100 000 >100 000 8.6 .sup.1Amount of MgO dosed.

Example 5: Three-Challenge Preservation Test

[0235] The indicated amounts of ingredients were introduced into 50 g samples of calcium carbonate slurry (77.6 wt.-% solids content). As antimicrobial agent a commercial available blend consisting of 2.5 wt.-% methylisothiazolinone (MIT, CAS No. 2682-20-4) 2.5 wt.-% benzisothiazolinone (BIT, CAS No. 2634-33-5), 8.5 wt.-% bronopol (BrnP, CAS No. 52-51-7) antimicrobial actives, based on aqueous content of the commercial antimicrobial agent, was dosed. Pure MgO was dosed from a suspension containing water and MgO with a purity of 99.7 wt.-% MgO based on total mineral content at 4 mol MgO/l suspension (corresponds to 161.24 g MgO/or 161.73 g minerals/l).

[0236] Burnt dolomite was dosed as ground powder at 625 ppm based on the aqueous content of the slurry. Half burnt dolomite was dosed as ground powder at 1 250 ppm based on aqueous content of the slurry.

[0237] In parallel, control samples of calcium carbonate slurry were prepared in absence of any additives. All samples were incubated to reach an equilibrium for 3 days at 30 C.

[0238] All samples were then inoculated three times with 1 mL of adapted mixture culture consisting of Comomonas aquatic, Pseudomonas stutzeri, Thauera mechernichensis, Brevundimonas intermedia, Rhizobium radiobacter, Spingobium yanoikuyae, Caldimonas sp., and Hydrogenophaga sp. After each inoculation, the samples were incubated at 30 C. for 24 hours. Thereafter, a 1:10 dilution of an aliquot was prepared in phosphate buffered saline (PBS) and was plated on TSA plates. These plates were incubated at 30 C. and analysed after 5 days. The first inoculation was performed 3 days, the second inoculation 5 days and the third inoculation 10 days after addition of ingredients.

[0239] The results of the inoculations are outlined in Table 3 below.

TABLE-US-00003 TABLE 3 Three-challenge Preservation test with a complex bacterial mixture as inoculum in slurry with different amounts of MgO, CaO and/or Biocide (BIT, MIT, Bronopol mixture) as antimicrobial agent. Additives in ppm per aqueous content Counts after Inoculation Dosage [cfu/ml] Biocide.sup.2 Dolomite Dolomite MgO.sup.1 CaO.sup.1 First Second Third 1 0 0 None 0 0 >100 000 >100 000 >100 000 3 300 0 None 0 0 >100 000 >100 000 >100 000 4 900 0 None 0 0 <100 200 500 5 0 625 Burnt 242 345 >100 000 >100 000 >100 000 7 300 625 Burnt 242 345 5 600 1 000 >10 000 8 900 625 Burnt 242 345 <100 <100 <100 9 0 1 250 Half burnt 363 0 >100 000 >100 000 >100 000 11 300 1 250 Half burnt 363 0 10 000 >10 000 >100 000 12 900 1 250 Half burnt 363 0 <100 <100 <100 .sup.1Amount of MgO and CaO dosed via the dolomite indicated. .sup.2Biocide consisted of 2.5 wt.-% MIT, 2.5 wt.-% BIT and 8.5 wt.-% bronopol

Example 6: Single-Challenge Preservation Test

[0240] The indicated amounts of ingredients were introduced into 3 ml samples of calcium carbonate slurry (77.8 wt.-% solids content). As antimicrobial agents, 2-phenylphenol (OPP, CAS No. 90-43-7) was dosed as 45% (w/w) solution, bronopol (BrnP, CAS NO. 52-51-7) was dosed as a 30% (w/w) solution and sodium pyrithione (NaPy, CAS NO. 3811-73-2) was dosed as a 40% (w/w) solution. Pure MgO was dosed as a suspension containing water and MgO with a purity of 99.7 wt.-% MgO based on the total mineral content at 2 mol MgO/l suspension (corresponds to 80.6 g MgO/ or 80.84 g minerals/l). In parallel, control samples of calcium carbonate slurry were prepared in absence of any additives. All samples were incubated to reach an equilibrium for at least 2 h at room temperature.

[0241] All samples were then inoculated once with 0.02 mL of an overnight culture of the indicated bacteria. All samples were incubated at 30 C. for 24 h. After incubation colony forming units (cfu) per plate (cfu/plate) were determined as described above. As Minimal inhibitory concentration (MIC) of a given MgO concentration for a bacterial strain, the lowest concentration of biocide where the bacterial concentration dropped below 100 cfu/plate, was defined.

[0242] The results of the inoculations are outlined in Tables 4, 5, 6 and 7 below.

TABLE-US-00004 TABLE 4 cfu/plate of a Single-challenge Preservation test after inoculation with E. coli as inoculum in slurry with different amounts of MgO and/or sodium pyrithione (NaPyr) as antimicrobial agent. NaPyr.sup.1 MgO.sup.2 Counts Inoculum ppm ppm cfu/plate 1 E. coli 300 0 >1 000 2 E. coli 150 3 000 3 3 E. coli 150 1 500 78 4 E. coli 150 750 >100 5 E. coli 150 0 >1 000 6 E. coli 30 3 000 >100 7 E. coli 30 1 500 >100 8 E. coli 30 750 >1 000 9 E. coli 30 0 >1 000 10 E. coli 0 3 000 >1 000 11 E. coli 0 1 500 >1 000 12 E. coli 0 750 >1 000 13 E. coli 0 0 >1 000 .sup.1Amount of sodium pyrithione dosed in ppm. .sup.2Amount of MgO dosed in ppm.

TABLE-US-00005 TABLE 5 cfu/plate of a Single-challenge Preservation test after inoculation with E. coli as inoculum in slurry with different amounts of MgO and/or orthophenylphenol (OPP) as antimicrobial agent. OPP.sup.1 MgO.sup.2 Counts Inoculum ppm ppm cfu/plate 1 E. coli 150 3 000 2 2 E. coli 150 1 500 >100 3 E. coli 150 750 >100 4 E. coli 150 0 >1 000 5 E. coli 0 3 000 >1 000 6 E. coli 0 1 500 >1 000 7 E. coli 0 750 >1 000 8 E. coli 0 0 >1 000 .sup.1Amount of orthophenylphenol dosed in ppm. .sup.2Amount of MgO dosed in ppm

TABLE-US-00006 TABLE 6 cfu/plate of a Single-challenge Preservation test after inoculation with S. aureus as inoculum in slurry with different amounts of MgO and/or sodium pyrithione (NaPyr) as antimicrobial agent. NaPyr.sup.1 MgO.sup.2 Counts Inoculum ppm ppm cfu/plate 1 S. aureus 300 0 >1 000 2 S. aureus 300 750 24 3 S. aureus 300 1 500 0 4 S. aureus 300 3 000 0 5 S. aureus 300 6 000 0 6 S. aureus 300 9 000 0 7 S. aureus 0 0 >1 000 8 S. auerus 0 750 >1 000 9 S. aureus 0 1 500 >1 000 10 S. aureus 0 3 000 >1 000 11 S. aureus 0 6 000 >1 000 12 S. aureus 0 9 000 >1 000 .sup.1Amount of sodium pyrithione dosed in ppm. .sup.2Amount of MgO dosed in ppm.

TABLE-US-00007 TABLE 7 cfu/plate of a Single-challenge Preservation test after inoculation with S. aureus as inoculum in slurry with different amounts of MgO and/or bronopol (BrnP) as antimicrobial agent. BrnP.sup.1 MgO.sup.2 Counts Inoculum ppm ppm cfu/plate 1 S. aureus 90 0 >1 000 2 S. aureus 90 750 >100 3 S. aureus 90 1 500 >100 4 S. aureus 90 3 000 6 5 S. aureus 90 6 000 14 6 S. aureus 90 9 000 1 7 S. aureus 30 0 >1 000 8 S. aureus 30 750 >1 000 9 S. aureus 30 1 500 >100 10 S. aureus 30 3 000 >100 11 S. aureus 30 6 000 >100 12 S. aureus 30 9 000 66 14 S. aureus 15 0 >1 000 15 S. aureus 15 750 >1 000 16 S. aureus 15 1 500 >100 17 S. aureus 15 3 000 20 18 S. aureus 15 6 000 >100 19 S. aureus 15 9 000 >100 20 S. aureus 0 0 >1 000 21 S. aureus 0 750 >1 000 22 S. aureus 0 1 500 >1 000 23 S. aureus 0 3 000 >1 000 24 S. aureus 0 6 000 >1 000 25 S. aureus 0 9 000 >1 000 .sup.1Amount of Bronopol dosed in ppm. .sup.2Amount of MgO dosed in ppm.

Example 7: Other Magnesium Sources

[0243] The indicated amounts of ingredients were introduced into 3 ml samples of calcium carbonate slurry (77.8 wt.-% solids content). As antimicrobial agent sodium pyrithione (NaPy, CAS NO. 3811-73-2) was dosed as a 40% (w/w) solution. Salts (MgO, MgCl.sub.2 or MgSO.sub.4) with a purity of >99%, were dosed from a suspension or solution. In parallel, control samples of calcium carbonate slurry were prepared in absence of any additives. All samples were incubated to reach an equilibrium for at least 2 h at room temperature.

[0244] All samples were then inoculated once with 0.02 ml of overnight culture of the indicated bacteria. All samples were incubated at 30 C. for 24 h. After incubation colony forming units (cfu) per plate (cfu/plate) were determined as described above.

[0245] The results of the inoculations are outlined in Table 8 below.

TABLE-US-00008 TABLE 8 cfu/plate of a Single-challenge Preservation test after inoculation with E. coli as inoculum in slurry with different amounts of magnesium salts (MgO, MgCl.sub.2 or MgSO.sub.4) and sodium pyrithione (NaPyr) as antimicrobial agent. Counts Inocu- NaPyr.sup.1 MgX.sup.2 MgO.sup.2 MgCl.sub.2.sup.2 MgSO.sub.4.sup.2 cfu/ lum ppm mmol/l ppm ppm ppm plate 1 E. coli 150 0 0 0 0 >1 000 2 E. coli 150 18.6 750 >100 3 E. coli 150 18.6 1 261 >1 000 4 E. coli 150 18.6 746 >1 000 5 E. coli 150 37.2 1 500 78 6 E. coli 150 37.2 2 522 >1 000 7 E. coli 0 0 0 0 0 >1 000 8 E. coli 0 18.6 750 >1 000 9 E. coli 0 18.6 1 261 >1 000 10 E. coli 0 18.6 746 >1 000 11 E. coli 0 37.2 1 500 >1 000 12 E. coli 0 37.2 2 522 >1 000 .sup.1Amount of sodium pyrithione dosed. .sup.2Amount of Magnesium salt dosed (MgO, MgSO.sub.4 or MgCl.sub.2) dosed.

Example 8: Curative Test with Three Post-Treatment Challenges

[0246] A natural contaminated industrial sample of calcium carbonate slurry (64.48 wt.-% solids content) was received to perform a curative treatment with preservation (i.e. removal of bacterial contamination and preservation from recontamination). This slurry had a particle size distribution of d.sub.50=1.6 m and 65 wt.-% of all particles have a particle size of <2 m. The indicated amounts of ingredients were introduced into 50 g samples of calcium carbonate slurry. As antimicrobial agent a commercial available blend consisting of 2.5 wt.-% methylisothiazolinone (MIT, CAS No. 2682-20-4) 2.5 wt.-% benzisothiazolinone (BIT, CAS No. 2634-33-5), 8.5 wt.-% bronopol (BrnP, CAS No. 52-51-7) antimicrobial actives was dosed.

[0247] Pure MgO was dosed from a suspension containing water and MgO with a purity of 99.7 wt.-% MgO based on the total mineral content at 4 mol MgO/l suspension (corresponds to 161.24 g MgO/l or 161.73 g minerals/l).

[0248] Half burnt dolomite was dosed as from a suspension containing water and 29 wt.-% MgO based on the total mineral content at 2 mol MgO/l suspension (corresponds to 80.62 g MgO/l or 278 g minerals/l).

[0249] In parallel, control samples of calcium carbonate slurry were prepared in absence of any additives. All samples were incubated for 24 h at 30 C. Thereafter, a 1:10 dilution of an aliquot was prepared in phosphate buffered saline (PBS) and was plated on TSA plates to enumerate the cfu/ml after curative treatment. These plates were incubated at 30 C. and analysed after 3 days.

[0250] All samples were then inoculated three times with 1 ml of untreated contaminated slurry to determine the preservation efficiency. After each inoculation of these three post-treatment challenges, the samples were incubated at 30 C. for 24 h. Thereafter, a 1:10 dilution of an aliquot was prepared in phosphate buffered saline (PBS) and was plated on TSA plates. These plates were incubated at 30 C. and analysed after 3 days. The first inoculation was performed 4 days, the second inoculation 7 days and the third inoculation 18 days after addition of ingredients.

[0251] Among the bacteria present in the contaminated slurry that produced cfu, the following species were identified: Staphylococcus capitis, Micrococcus luteus, Bacillus halodurans, Ochrobactrum sp., Ochrobactrum tritici and Kocuria rhizophila. Species were identified by Bruker Daltonics MALDI Biotyper (Billerica, Mass., USA).

[0252] The results of the inoculations are outlined in Table 9 below.

TABLE-US-00009 TABLE 9 Curative test with three post-treatment challenges of a slurry containing a natural occurring bacterial mixture. The slurry was treated with different amounts of MgO and/or Biocide (BIT, MIT, Bronopol mixture) as antimicrobial agent. Additives in ppm per Counts after Inoculation aqueous content cfu/ml Biocide.sup.2 MgO.sup.1 Mineral Curative First Second Third 1 0 0 None >100 000 >100 000 >100 000 >100 000 2 0 1 500 MgO >100 000 >100 000 >100 000 >100 000 3 0 3 000 MgO >100 000 >100 000 >100 000 >100 000 4 0 1 500 Half burnt >100 000 >100 000 >100 000 >100 000 5 0 3 000 Half burnt >10 000 >100 000 >100 000 9 000 6 50 0 None >10 000 1 200 3 800 7 800 7 50 1 500 MgO <100 <100 300 600 8 50 3 000 MgO <100 <100 6 000 <100 9 50 1 500 Half burnt <100 <100 <100 <100 10 50 3 000 Half burnt <100 100 600 <100 11 100 0 None >10 000 <100 2 900 3 400 12 100 1 500 MgO <100 <100 <100 <100 13 100 3 000 MgO <100 <100 <100 <100 14 100 1 500 Half burnt <100 100 <100 <100 15 100 3 000 Half burnt <100 <100 300 <100 16 200 0 None 6 600 100 1 600 3 400 17 200 1 500 MgO <100 <100 <100 <100 18 200 3 000 MgO <100 <100 <100 <100 19 200 1 500 Half burnt <100 100 100 <100 20 200 3 000 Half burnt <100 <100 <100 <100 .sup.1Amount of MgO dosed via the mineral indicated. .sup.2Biocide consisted of 2.5 wt.-% MIT, 2.5 wt.-% BIT and 8.5 wt.-% bronopol

Example 9: Three-Challenge Preservation Test with Slurry Samples Prepared Combining all Minerals Prior Grinding

[0253] Slurry samples without additive (75 wt.-% solids content) were used as control. As pH-control, slurries without additives were adjusted to pH of 10.0+/0.1 using 2 mol/l NaOH in water.

[0254] Slurry samples (75.4 wt.-% solids content) containing MgO were prepared by adding the MgO with 97.7 wt.-% MgO content at 1 000 ppm based on total mineral content (dry weight) prior grinding. After grinding the pH was 10.52. The pH was adjusted by adding slurry without additive to a pH of 10.0+/0.1.

[0255] Slurry samples (75.6 wt.-% solids content) containing half burnt dolomite were prepared by adding 3 000 ppm half burnt dolomite based on total mineral content (dry weight) to the carbonates prior grinding. The half burnt dolomite contained 29 wt.-% MgO. After grinding the pH was 10.8. The pH was adjusted by adding slurry without additive to a pH of 10.0+/0.1.

[0256] After pH adjustments, antimicrobial agent was added to 50 g samples of the slurry samples indicated. As antimicrobial agent methylisothiazolinone (MIT, CAS No. 2682-20-4) was dosed as 20% (w/w) solution. As another antimicrobial agent a commercial available blend consisting of 2.5 wt.-% methylisothiazolinone (MIT, CAS No. 2682-20-4) 2.5 wt.-% benzisothiazolinone (BIT, CAS No. 2634-33-5), 8.5 wt.-% bronopol (BrnP, CAS No. 52-51-7) antimicrobial actives was dosed.

[0257] In parallel, control samples of calcium carbonate slurry were prepared in absence of any antimicrobial agents. All samples were incubated to reach an equilibrium for 3 days at 30 C.

[0258] All samples were then inoculated three times with 1 mL of adapted mixture culture consisting of Comomonas aquatic, Pseudomonas stutzeri, Thauera mechernichensis, Brevundimonas intermedia, Rhizobium radiobacter, Spingobium yanoikuyae, Caldimonas sp., and Hydrogenophaga sp. After each inoculation, the samples were incubated at 30 C. for 24 hours. Thereafter, a 1:10 dilution of an aliquot was prepared in phosphate buffered saline (PBS) and was plated on TSA plates. These plates were incubated at 30 C. and analysed after 2 days. The first inoculation was performed 3 days, the second inoculation 5 days and the third inoculation 10 days after addition of ingredients.

[0259] The results of the inoculations are outlined in Table 10 below.

TABLE-US-00010 TABLE 10 Three-challenge Preservation test with a complex bacterial mixture as inoculum in slurries with pH adjusted to 10 +/ 0.1 using MgO, half burnt dolomite or NaOH and/or Biocide (BIT, MIT, Bronopol mixture) as antimicrobial agent. Additives in ppm per Counts after Inoculation aqueous content cfu/ml Additive.sup.1 pH.sup.2 Biocide.sup.3 First Second Third 1 None 9.15 0 >100 000 >100 000 >100 000 2 None 860 <100 9 700 >10 000 3 Half burnt 10.1 0 >100 000 >100 000 >100 000 4 Half burnt 860 <100 3 200 <100 5 MgO 9.96 0 >100 000 >100 000 >100 000 6 MgO 860 <100 1 300 <100 7 NaOH 10.02 0 >100 000 >100 000 >100 000 8 NaOH 860 700 2 400 >10 000 .sup.1Additive used to adjust pH to 10 +/ 0.1 .sup.2pH prior addition of MIT. .sup.3Biocide consisted of 2.5 wt.-% MIT, 2.5 wt.-% BIT and 8.5 wt.-% bronopol

Example 10: Single-Challenge Preservation Test in Buffered Aqueous System

[0260] Phosphate buffered saline (PBS, pH 7.4) without minerals was used as control without pH adjustment. Samples of PBS were adjusted to pH 10.1+/0.1 using either 2 mol/l NaOH in water or pure MgO or burnt dolomite. MgO had a purity of 99.7 wt.-% based on total mineral content and was dosed as 4 mol MgO/l suspension (corresponds to 161.24 g MgO/l or 161.73 g minerals/l). Burnt dolomite with an MgO content of 38.7 wt.-% was dosed from a suspension at 2 mol MgO/l suspension (corresponds to 80.6 g MgO/l or 208.3 g burnt dolomite/l).

[0261] After pH adjustments, antimicrobial agent was added to 10 ml samples of the PBS samples indicated. As antimicrobial agent methylisothiazolinone (MIT, CAS No. 2682-20-4) was dosed as 20% (w/w) solution. As another antimicrobial agent a commercial available blend consisting of 2.5 wt.-% methylisothiazolinone (MIT, CAS No. 2682-20-4) 2.5 wt.-% benzisothiazolinone (BIT, CAS No. 2634-33-5), 8.5 wt.-% bronopol (BrnP, CAS No. 52-51-7) antimicrobial actives was dosed.

[0262] For the preparation of the inoculum, overnight cultures of the bacteria Comomonas aquatic, Pseudomonas stutzeri, Thauera mechernichensis, Brevundimonas intermedia, Rhizobium radiobacter, Spingobium yanoikuyae, Caldimonas sp., and Hydrogenophaga sp. were prepared and combined in equal ratio (i.e. 0.1 ml of each overnight culture. The mixture was further diluted 1:100 in TSB. Of the dilution 0.01 ml was used to inoculate 0.9 ml of the PBS sample. The inoculated samples were incubated for 24 h at 30 C. After incubation, 0.1 ml was directly plated onto TSA plates and the plates were further incubated at 30 C. for 3 days prior enumeration of the cfu/ml. Counts from 10 000 cfu/ml to 99 999 cfu/ml are reported as >10 000 cfu/ml and counts from 1 000 cfu/ml to 9 999 cfu/ml are reported as >1 000 cfu/ml

[0263] The results of the inoculations are outlined in Tables 11 and 12 below.

TABLE-US-00011 TABLE 11 Single-challenge Preservation test with a complex bacterial mixture as inoculum in PBS pH adjusted to 10 +/ 0.1 using MgO or NaOH and/or MIT as antimicrobial agent. Additives in ppm per Counts after aqueous content Inoculation Additive.sup.1 pH.sup.2 MIT [cfu/ml] 1 None 7.4 0 >10 000 2 None 10 >10 000 3 None 20 >10 000 4 None 40 >1 000 5 Burnt Dolomite 10.1 0 >1 000 6 Burnt Dolomite 10 240 7 Burnt Dolomite 20 80 8 Burnt Dolomite 40 30 9 MgO 10.2 0 >1 000 10 MgO 10 20 11 MgO 20 40 12 MgO 40 <10 13 NaOH 10.1 0 >10 000 14 NaOH 10 >10 000 15 NaOH 20 >10 000 16 NaOH 40 980 .sup.1Additive used to adjust pH of PBS to 10 +/ 0.1 .sup.2pH prior addition of MIT.

TABLE-US-00012 TABLE 12 Single-challenge Preservation test with a complex bacterial mixture as inoculum in PBS pH adjusted to 10 +/ 0.1 using MgO or NaOH and/or Biocide (BIT, MIT, Bronopol mixture) as antimicrobial agent. Additives in ppm per Counts after aqueous content Inoculation Additive.sup.1 pH.sup.2 Biocide.sup.3 [cfu/ml] 1 None 7.4 0 >10 000 2 None 25 >10 000 3 None 50 >10 000 4 None 100 >1 000 5 Burnt Dolomite 10.1 0 >1 000 6 Burnt Dolomite 25 260 7 Burnt Dolomite 50 140 8 Burnt Dolomite 100 70 9 MgO 10.2 0 >1 000 10 MgO 25 220 11 MgO 50 80 12 MgO 100 30 13 NaOH 10.1 0 >10 000 14 NaOH 25 >10 000 15 NaOH 50 >1 000 16 NaOH 100 >1 000 .sup.1Additive used to adjust pH of PBS to 10 +/ 0.1 .sup.2pH prior addition of MIT. .sup.3Biocide consisted of 2.5 wt.-% MIT, 2.5 wt.-% BIT and 8.5 wt.-% bronopol